MicroRNAs (miRNAs) have demonstrated great promise as biomarkers for investigation of cancer development and progression and for early detection, yet these small molecules have not been adopted into early diagnostics for clinical practice because of challenging analytical aspects in the lab. Microarray-based technology, Northern blotting, sequencing, and qRT-PCR analyses are often employed, involving elaborate, time-consuming and expensive processes that require special laboratory equipment. There is a need to develop alternative molecular assay strategies that may offer more advantages over conventional methods and have the potential to enhance research in the areas of early detection and screening, and clinical diagnosis. Although miRNAs related to gastrointestinal (GI) cancer will be used as the model system, the proposed molecular analysis technology will be applicable generally to other types of cancers and other miRNA probes. We will develop a novel molecular analysis technology based on the Inverse Molecular Sentinel (iMS) scheme using surface-enhanced Raman scattering (SERS) detection that will move miRNA testing from an RT-PCR lab-based based assay scheme to a molecular analysis and testing technology that is simpler, faster and more cost effective. The specific aims of this project are: (1) Develop the new iMS molecular analysis method for multiplex detection of molecular biomarkers, miRNAs. Due to the highly specific and narrow SERS peaks, we will use the iMS technique to simultaneously detect multiple microRNA sequences as a simple and rapid multiplex screening tool for esophageal cancer. The analytical features of merit (specificity, sensitivity, probe stability) of the new molecular analysis technology will be investigated in detail. (2) Apply and evaluate the iMS molecular analysis method for diagnosis and investigation of GI cancer development and progression using clinical samples. We will demonstrate the usefulness of the iMS technique to detect miRNAs in peripheral blood samples from patients with known cancer diagnosis and from controls. In the clinical cohort, RT-PCR will be used as the gold standard for diagnosis, and nanoprobes will be tested against RT-PCR for diagnostic accuracy. The iMS biosensing system has the potential to be faster and more efficient than currently available systems, and is capable of providing great sensitivity with higher levels of specificity and multiplexing capabilities to screen microRNAs associated with Barrett's esophagus and esophageal cancer. In conclusion, the proposed molecular analysis technology represents a major innovation that has great transformative potential in cancer research, diagnostics and screening. The iMS approach is a `homogenous' assay (i.e., requiring no sample removal, and thus no microfluids). Also, the targets do not need to be labeled, thus reducing cost and complexity. The advantages of the novel molecular analysis technology would allow detection of multiple miRNA biotargets for research on cancer development and progression, and ultimately enable its future clinical translation to render a faster and more accurate diagnosis at point-of-care settings.